Astragaloside IV attenuates renal tubule injury in DKD rats via suppression of CD36-mediated NLRP3 inflammasome activation.

Background: In recent years, diabetic kidney disease (DKD) has emerged as a prominent factor contributing to end-stage renal disease. Tubulointerstitial inflammation and lipid accumulation have been identified as key factors in the development of DKD. Earlier research indicated that Astragaloside IV (AS-IV) reduces inflammation and oxidative stress, controls lipid accumulation, and provides protection to the kidneys. Nevertheless, the mechanisms responsible for its protective effects against DKD have not yet been completely elucidated. Purpose: The primary objective of this research was to examine the protective properties of AS-IV against DKD and investigate the underlying mechanism, which involves CD36, reactive oxygen species (ROS), NLR family pyrin domain containing 3 (NLRP3), and interleukin-1ß (IL-1ß). Methods: The DKD rat model was created by administering streptozotocin along with a high-fat diet. Subsequently, the DKD rats and palmitic acid (PA)-induced HK-2 cells were treated with AS-IV. Atorvastatin was used as the positive control. To assess the therapeutic effects of AS-IV on DKD, various tests including blood sugar levels, the lipid profile, renal function, and histopathological examinations were conducted. The levels of CD36, ROS, NLRP3, Caspase-1, and IL-1ß were detected using western blot analysis, PCR, and flow cytometry. Furthermore, adenovirus-mediated CD36 overexpression was applied to explore the underlying mechanisms through in vitro experiments. Results: In vivo experiments demonstrated that AS-IV significantly reduced hyperglycemia, dyslipidemia, urinary albumin excretion, and serum creatinine levels in DKD rats. Additionally, it improved renal structural abnormalities and suppressed the expression of CD36, NLRP3, IL-1ß, TNF-α, and MCP-1. In vitro experiments showed that AS-IV decreased CD36 expression, lipid accumulation, and lipid ROS production while inhibiting NLRP3 activation and IL-1ß secretion in PA-induced HK-2 cells. Conclusion: AS-IV alleviated renal tubule interstitial inflammation and tubule epithelial cell apoptosis in DKD rats by inhibiting CD36-mediated lipid accumulation and NLRP3 inflammasome activation.

(+)-Catechin ameliorates diabetic nephropathy injury by inhibiting endoplasmic reticulum stress-related NLRP3-mediated inflammation.

Endoplasmic reticulum (ER) stress and chronic sterile inflammation are associated with the pathogenesis of diabetic nephropathy (DN). Catechins are natural polyphenolic compounds found in green tea that possess some health benefits. However, whether (+)-catechin can reduce tubular injury in DN by regulating ER stress and NLRP3-associated inflammation remains uncertain. This study examined the effects of (+)-catechin on streptozotocin (STZ)-induced diabetic mice and on palmitic acid (PA)-treated HK-2 cells. In vivo, a DN mouse model was generated by injecting STZ. The biochemical indicators of serum and urine, as well as renal histopathology and ultrastructure were analysed. To predict the mechanisms associated with (+)-catechin, network pharmacology and molecular docking were used. Finally, quantitative real-time PCR (qPCR), western blot analysis and immunofluorescence analysis were performed to measure the mRNA and protein expressions of specific targets in the renal tissue of DN mice and PA-treated HK-2 cells to validate the predicted results. (+)-Catechin significantly ameliorated renal function and pathological changes associated with tubular injury by inhibiting ER stress by downregulating of GRP78, PEAK, CHOP, ATF6 and XBP1. In addition, (+)-catechin inhibited renal inflammation by suppressing NLRP3 associated inflammation, which was characterized by the downregulation of NLRP3, ASC, AIM2, Caspase1, IL-1ß and IL-18 in DN mice and PA-treated HK-2 cells. Collectively, these findings suggested that (+)-catechin exerted a renoprotective effect against DN by inhibiting ER stress and NLRP3-related inflammation to ameliorate tubular injury, suggesting the therapeutic potential of (+)-catechin.

Development and validation of a prediction model for all-cause mortality in maintenance dialysis patients: a multicenter retrospective cohort study.

BACKGROUND: The mortality risk varies considerably among individual dialysis patients. This study aimed to develop a user-friendly predictive model for predicting all-cause mortality among dialysis patients. METHODS: Retrospective data regarding dialysis patients were obtained from two hospitals. Patients in training cohort (N = 1421) were recruited from the Fifth Affiliated Hospital of Sun Yat-sen University, and patients in external validation cohort (N = 429) were recruited from the First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine. The follow-up endpoint event was all-cause death. Variables were selected by LASSO-Cox regression, and the model was constructed by Cox regression, which was presented in the form of nomogram and web-based tool. The discrimination and accuracy of the prediction model were assessed using C-indexes and calibration curves, while the clinical value was assessed by decision curve analysis (DCA). RESULTS: The best predictors of 1-, 3-, and 5-year all-cause mortality contained nine independent factors, including age, body mass index (BMI), diabetes mellitus (DM), cardiovascular disease (CVD), cancer, urine volume, hemoglobin (HGB), albumin (ALB), and pleural effusion (PE). The 1-, 3-, and 5-year C-indexes in the training set (0.840, 0.866, and 0.846, respectively) and validation set (0.746, 0.783, and 0.741, respectively) were consistent with comparable performance. According to the calibration curve, the nomogram predicted survival accurately matched the actual survival rate. The DCA showed the nomogram got more clinical net benefit in both the training and validation sets. CONCLUSIONS: The effective and convenient nomogram may help clinicians quantify the risk of mortality in maintenance dialysis patients.

Emodin alleviates CRS4-induced mitochondrial damage via activation of the PGC1α signaling.

Cardiorenal syndrome type 4 (CRS4), a progressive deterioration of cardiac function secondary to chronic kidney disease (CKD), is a leading cause of death in patients with CKD. In this study, we aimed to investigate the cardioprotective effect of emodin on CRS4. C57BL/6 mice with 5/6 nephrectomy and HL-1 cells stimulated with 5% CKD mouse serum were used for in vivo and in vitro experiments. To assess the cardioprotective potential of emodin, we employed a comprehensive array of methodologies, including echocardiography, tissue staining, immunofluorescence staining, biochemical detection, flow cytometry, real-time quantitative PCR, and western blot analysis. Our results showed that emodin exerted protective effects on the function and structure of the residual kidney. Emodin also reduced pathologic changes in the cardiac morphology and function of these mice. These effects may have been related to emodin-mediated suppression of reactive oxygen species production, reduction of mitochondrial oxidative damage, and increase of oxidative metabolism via restoration of PGC1α expression and that of its target genes. In contrast, inhibition of PGC1α expression significantly reversed emodin-mediated cardioprotection in vivo. In conclusion, emodin protects the heart from 5/6 nephrectomy-induced mitochondrial damage via activation of the PGC1α signaling. The findings obtained in our study can be used to develop effective therapeutic strategies for patients with CRS4.

Metal dimer nanojunction-magnetic material composites for magnetic field sensing.

Noble metal nanocrystals are used as high sensitivity optoelectronic sensors, such as surface-enhanced Raman scattering, SERS. The sensing performance of metal nanocrystals can be further improved by forming dimer nanojunctions with strong "plasmonic coupling". Since the strength of "plasmonic coupling" is highly sensitive to the sub-nanoscale spacing between plasmonic nanocrystals in nanojunctions, nanojunctions can be used to detect external stimuli that can change the spacing of nanocrystals in the nanojunction and thus change the sensitivity of the Raman scattering spectrum. Here, we utilize this principle to detect the direction and strength of an external magnetic field (MF) using dimer nanojunctions surrounded by magnetic materials as a sensing platform. The results reveal that the changes in nanocrystal spacing in the nanojunction are caused by the rearrangement of the magnetic material under an external MF, which strongly depends on the interaction between the magnetic material and the ligands on the nanocrystal surface and the steric repulsion generated by the ligand configuration on the nanocrystal surface. Compared with the Raman spectrum without an external MF, the enhancement factors of the Raman scattering spectrum under an external MF can reach up to ∼900%, which makes dimer nanojunctions with magnetic materials suitable for "magnetic field" sensing applications.

The estimated glomerular filtration rate was U-shaped associated with abdominal aortic calcification in US adults: findings from NHANES 2013-2014.

Objectives: The high incidence of abdominal aortic calcification (AAC) is well-documented in individuals with severe renal function decline. However, there is limited research on the historical relationship between estimated glomerular filtration rate (eGFR) and the risk of AAC occurrence in the general population undergoing routine medical examinations. The main objective of this study was to investigate the historical relationship between eGFR and AAC in the general population of the United States. Methods: We performed a cross-sectional study using the National Health and Nutrition Examination Survey 2013-2014 database. Weighted multivariate linear regression models were used to estimate the associations of eGFR with AAC score. Smooth curve fitting and two-piecewise linear regression were employed to explore the potential non-linear relationship. Results: A total of 2,978 participant (48.22% were male) aged 40-80 years were included in this study. The fully-adjusted model demonstrated a negative correlation between eGFR and AAC score (ß = -0.015, 95% CI: -0.023 to -0.006). However, when applying the smooth curve fitting method, a U-shaped relationship was identified, and the inflection point was calculated at 76.43 ml/min/1.73 m2 using the two-piecewise linear regression model. Conclusions: There was a U-shaped association between eGFR and AAC score in general US adults, with an inflection point at about 76.43 ml/min/1.73 m2.

The association between socioeconomic status and health-related quality of life among young and middle-aged maintenance hemodialysis patients: multiple mediation modeling.

Objective: To explore the relationship between socioeconomic status (SES), illness perception, social functioning, and health-related quality of life (HRQoL) of young and middle-aged maintenance hemodialysis (MHD) patients and the internal mechanism of action. Design: A multicenter cross-sectional study. Methods: An aggregate of 332 young and middle-aged MHD patients were enrolled from hemodialysis centers in four general hospitals in Guangzhou, Guangdong, China, from June to December 2022. The questionnaires used included one for general demographic data, the Brief Illness Perception Questionnaire (BIPQ), Social Dysfunction Screening Scale (SDSS), and the 12-item Short Form Health Survey (SF-12). Results: Both SES and HRQoL were negatively correlated with illness perception and social functioning, respectively. SES was positively correlated with HRQoL. Illness perception was positively correlated with social functioning. The indirect effects of illness perception and social functioning on the relationship between SES and HRQoL were 0.33 and 0.31, making up 41.06% and 38.91% of the sum. The chain indirect effect of illness perception and social functioning was 0.10, making up 12.59% of the total effect, while gender did not play a moderating role. Conclusion: Illness perception and social functioning may independently and accumulatively mediate the association between SES and HRQoL. Nurses should consider developing individual intervention program for young and middle-aged MHD patients with low SES, focusing on establishing targeted counseling and health education strategies corresponding to illness perception and social functioning to help patients improve their HRQoL.

Potential role of microRNA-503 in Icariin-mediated prevention of high glucose-induced endoplasmic reticulum stress.

BACKGROUND: Dysregulated microRNA (miRNA) is crucial in the progression of diabetic nephropathy (DN). AIM: To investigate the potential molecular mechanism of Icariin (ICA) in regulating endoplasmic reticulum (ER) stress-mediated apoptosis in high glucose (HG)-induced primary rat kidney cells (PRKs), with emphasis on the role of miR-503 and sirtuin 4 (SIRT4) in this process. METHODS: Single intraperitoneal injection of streptozotocin (65 mg/kg) in Sprague-Dawley rats induce DN in the in vivo hyperglycemic model. Glucose-treated PRKs were used as an in vitro HG model. An immunofluorescence assay identified isolated PRKs. Cell Counting Kit-8 and flow cytometry analyzed the effect of ICA treatment on cell viability and apoptosis, respectively. Real-time quantitative polymerase chain reaction and western blot analyzed the levels of ER stress-related proteins. Dual luciferase analysis of miR-503 binding to downstream SIRT4 was performed. RESULTS: ICA treatment alleviated the upregulated miR-503 expression in vivo (DN) and in vitro (HG). Mechanistically, ICA reduced HG-induced miR-503 overexpression, thereby counteracting its function in downregulating SIRT4 levels. ICA regulated the miR-503/SIRT4 axis and subsequent ER stress to alleviate HG-induced PRKs injury. CONCLUSION: ICA reduced HG-mediated inhibition of cell viability, promotion of apoptosis, and ER stress in PRKs. These effects involved regulation of the miR-503/SIRT4 axis. These findings indicate the potential of ICA to treat DN, and implicate miR-503 as a viable target for therapeutic interventions in DN.

Astragaloside IV attenuates myocardial dysfunction in diabetic cardiomyopathy rats through downregulation of CD36-mediated ferroptosis.

Diabetic cardiomyopathy (DCM), one of the major complications of type 2 diabetes, is a leading cause of heart failure and death in advanced diabetes. Although there is an association between DCM and ferroptosis in cardiomyocytes, the internal mechanism of ferroptosis leading to DCM development remains unknown. CD36 is a key molecule in lipid metabolism that mediates ferroptosis. Astragaloside IV (AS-IV) confers various pharmacological effects such as antioxidant, anti-inflammatory, and immunomodulatory. In this study, we demonstrated that AS-IV was able to recover the dysfunction of DCM. In vivo experiments showed that AS-IV ameliorated myocardial injury and improved contractile function, attenuated lipid deposition, and decreased the expression level of CD36 and ferroptosis-related factors in DCM rats. In vitro experiments showed that AS-IV decreased CD36 expression and inhibited lipid accumulation and ferroptosis in PA-induced cardiomyocytes. The results demonstrated that AS-IV decreased cardiomyocyte injury and myocardial dysfunction by inhibiting ferroptosis mediated by CD36 in DCM rats. Therefore, AS-IV regulated the lipid metabolism of cardiomyocytes and inhibited cellular ferroptosis, which may have potential clinical value in DCM treatment.

Detection of Cancer Marker Flap Endonuclease 1 Using One-Pot Transcription-Powered Clustered Regularly Interspaced Short Palindromic Repeat/Cas12a Signal Expansion.

As a critical functional protein in DNA replication and genome stability, flap endonuclease 1 (FEN1) has been considered a promising biomarker and druggable target for multiple cancers. We report here a transcription-powered clustered regularly interspaced short palindromic repeat (CRISPR)/Cas12a signal expansion platform for rapid and sensitive detection of FEN1. In this method, the probe cleavage by FEN1 generated a free 5' flap single-stranded DNA which could hybridize with the single-stranded T7 promoter-bearing template and trigger the extension. Then, the CRISPR guide RNA (crRNA) transcribed from the extended template activated the collateral DNase activity of Cas12a, releasing the fluorophore from the quenched DNA signal probe to report the FEN1 detection result. The high specificity for FEN1 was validated by comparing with other repair-relevant proteins. The limit of detection (LOD) could be as low as 0.03 mU, which is sensitive enough to detect the FEN1 activity in biological samples. In addition, the inhibition assay of FEN1 was also successfully achieved with this platform, proving its potential in inhibitor screening. In summary, this study provides a novel biosensor for FEN1 activity analysis and provides new insights into the development of CRISPR-based biosensors for non-nucleic acid targets.

Real-time fluorescence and colorimetric identification of bulbus fritillariae using recombinase assisted loop-mediated isothermal DNA amplification (RALA).

Bulbus Fritillariae (BF) is a kind of herbal medicine widely used in many countries including China, Japan, Korea, and so on. Among the known BF medicinal materials, Bulbus Fritillariae cirrhosae (BF cirrhosae) was reported to have the best curative effect. Due to the limited resources of BF cirrhosae, a lot of adulterants have emerged in the market, impairing the market order, resource development, and above all, clinical efficacy. Here, a novel nucleic acid amplification technique, Recombinase Assisted Loop-mediated isothermal DNA Amplification (RALA), was used to establish a real-time fluorescence isothermal molecular authentication method for five commonly used BF drugs. Moreover, this RALA-based assay can also be developed as a colorimetric detection method for on-site detection. Both real-time fluorescence and visual methods could detect as low as 0.1% genuine targets in the mixed samples. In summary, we report an isothermal detection system for five kinds of BF circulating in the market, providing a new choice for the molecular identification of BF drugs and showing promise in the laboratory testing as well as field identification of other herbal medicines.

Icariin Attenuation of Diabetic Kidney Disease Through Inhibition of Endoplasmic Reticulum Stress via G Protein-Coupled Estrogen Receptors.

Diabetic kidney disease (DKD) is the most common complication of diabetes mellitus and has become the primary cause of End-Stage Renal Disease (ESRD) globally. Icariin (ICA), an effective component extracted from Epimedium, has antiosteoporosis effect, antitumor effects, anti-ischemia effects, and other effects. In this study, a mouse DKD model was established, and Icariin solid nanoliposomes were administered to determine whether ICA had a protective effect on the renal function of DKD mice by regulating estrogen level and endoplasmic reticulum (ER) stress pathway. The results showed that the microalbumin/creatinine in urine, serum urea nitrogen, and CHOL in ICA cultured DKD mice significantly decreased, and mice nephropathy improved significantly. rat renal tubule epithelial cells were further tested, and the rat renal tubule epithelial cells were modeled by cultured cells with high glucose. The results showed that high glucose could promote the proliferation of renal tubular epithelial cells. Simultaneously, ICA can inhibit the proliferation of renal tubular epithelial cells and induce cell apoptosis. Furthermore, the expression of ER stress-related proteins IRE1 and XBP-1S was further detected. Additionally, to ICA intervention, a GPER antagonist (G-15) was added for intervention, the inhibitory effects of IRE1 and XBP-1S were reversed, and the ER stress pathway was activated. Cell experiments showed that ICA could promote GPER expression, while inhibiting GPER expression promoted the activation of ER stress pathway, and GPER expression was negatively correlated with ER stress protein expression. Therefore, the experiment proved that in DKD tissues, a high concentration of ICA can inhibit the ER stress response by promoting the expression of GPER, reducing the proliferation of diabetic nephropathy, and increasing the rate of tissue apoptosis.

Platycodin D regulates high glucose-induced ferroptosis of HK-2 cells through glutathione peroxidase 4 (GPX4).

Diabetic nephropathy (DN) is associated with inflammation. Platycodin D (PD) demonstrates anti-inflammatory activity. However, whether PD affects DN remains to be explored. Here, we aimed to discuss the role of PD in DN and its underlying mechanisms. High glucose (HG)-induced HK-2 cells were treated with PD, and cell viability was assessed using the Thiazolyl Blue Tetrazolium Bromide (MTT) assay. Ferroptosis-related factors such as lactate dehydrogenase (LDH) activity, lipid reactive oxygen species (ROS), iron (Fe2+) level, GSH level, and malondialdehyde (MDA) level were evaluated. Cell death was evaluated using the TUNEL assay. GPX4 expression was evaluated using Quantitative Real-time PCR (qRT-PCR) and Western blotting analysis. The results indicated that HG increased LDH activity, lipid ROS production, Fe2+ levels, and MDA levels and decreased GSH levels, suggesting that the HG condition induced ferroptosis. PD treatment inhibited ferroptosis in HG-induced cells, downregulated ACSL4 and TFR1 expression, and upregulated FTH-1 and SLC7A11 expression. PD reversed the effects of HG condition on cell death. Moreover, GPX4 expression was downregulated in HG-stimulated cells. Furthermore, we substantiated that PD suppressed ferroptosis by modulating GPX4 expression. In conclusion, PD inhibited ferroptosis in HG-induced HK-2 cells by upregulating GPX4 expression, suggesting that PD may be an effective drug for the clinical treatment of DN.

Quercetin alleviates chronic renal failure by targeting the PI3k/Akt pathway.

Chronic renal failure (CRF) threatens human health greatly and attracts worldwide concerns of health professionals in the public health sector. In our preliminary study, we found that Compound capsule (Shengqing Jiangzhuo Capsule, SQJZJN) had a significant therapeutic effect on CRF. Quercetin is one of the main components of this Compound capsule. In this study, we investigated the effect of Quercetin monomer on CRF and the regulation of PI3k/Akt pathway. Network pharmacology analysis methods were employed to analyze the SQJZJN/Quercetin/PIK3R1 network relationships. In this study, a CRF rat model was prepared using the gavage adenine solution method and detected the indicators of Creatinine (Cr), Blood Urea Nitrogen (BUN), and Uric Acid (UA). After treating the rat model with Quercetin and PIK3R1-interfering lentivirus, respectively, we observed the changes on the histological morphology of the kidney and detected apoptosis using TUNEL staining. Gene and protein expression associated with renal function were detected using qPCR, WB and immunofluorescence. Quercetin was identified as the main ingredient of SQJZJN by the network pharmacological screening and Quercetin at 1.5 and 3 g/(kg.d) concentrations could effectively alleviate the CRF symptoms, reduce the levels of Cr, BUN, and UA, and markedly inhibit cell apoptosis demonstrated by the intragastric administration. Furthermore, the protein expression of p-PI3K, p-AKT, NLRP3, caspase1, AQP1, and AQP2 in all groups was detected by immunofluorescence and western blot assays, indicating that Quercetin could reduce the expression of NLRP3, caspase1, p-PI3k, and p-Akt, and increase the expression of AQP1 and AQP2 in the renal tissues of CRF rats. Being labeled with biotin and incubated with the total protein extracted from kidney tissues, Quercetin could bind to PIK3R1. Following the PIK3R1 interference lentivirus was injected into the CRF model rats by tail vein, the CRF symptoms were effectively alleviated in the PIK3R1 interference group, consistent with the effect of Quercetin. Taken together, Quercetin, a major component of SQJZJN, might minimize renal fibrosis and apoptosis in CRF rats by inhibiting the PI3k/Akt pathway through targeting PIK3R1. By regulating AQP1 and AQP2, both water retention and toxin accumulation were reduced.

Patient and Graft Survival After Dual Kidney Transplantation With Marginal Donors in Comparison to Matched Control Groups.

BACKGROUND: Postmortal organ donor rates remain low in Germany, whereas donor age has been increasing considerably in the last decades. As a consequence of low donation rates older and more marginal donor kidneys are accepted for transplantation. However, procured kidneys from very old a/o marginal donors may be considered as not suitable for transplantation as a single organ and subsequently be discarded. However, dual transplantation of both kidneys from such donors may provide an opportunity to nevertheless use these organs for renal transplantation, thereby providing the twofold nephron mass as a single kidney transplantation. METHODS: We compared in this retrospective analysis the outcome of 10 recipients of a dual kidney transplantation (DKT) with 40 matched recipients of a single kidney transplantation (SKT). Recipients were matched for donor and recipient age (ie, a maximum age difference of ±10 years in a ratio of 1:4 for DKT vs SKT recipients). In addition, a second SKT control group of 10 SKT recipients being transplanted immediately before each DKT recipient with a kidney from a donor aged ≥65 years was used for comparison. All renal transplant recipients were observed for up to 3 years or until July 31, 2020. RESULTS: Mean donor and recipient age was 77.2 ± 4.6/75.1 ± 6.6/82.1 ± 7.9 and 66.4 ± 5.8/66.1 ± 6.0/64.8 ± 8.4 for SKT group 1/SKT group 2/DKT, respectively. Procurement serum creatinine concentrations were significantly higher in the DKT group in comparison to the SKT control group 1 (P = .019) as was the rate of transplant artery atherosclerosis (P = .021). Furthermore, Kidney Donor Profile Index, and Kidney Donor Risk Index were significantly higher (P = .0138/P = .064, and P < .001/P = .038) in the DKT group than in SKT group 1 and 2. Rates of acute rejection and delayed graft function were not significantly different between groups, though biopsy-proven acute rejection was numerically higher in the SKT groups. Patient survival and overall and death-censored graft survival rates were also not significantly different between groups, although they tended to be higher after DKT. CONCLUSIONS: DKT provides an opportunity to successfully use postmortal kidneys even from donors aged >80 years and a Kidney Donor Profile Index ≥95% for renal transplantation. DKT may thereby increase the available pool of donors to better serve patients with end-stage renal disease on the waiting list.

Sequence-specific and multiplex detection of COVID-19 virus (SARS-CoV-2) using proofreading enzyme-mediated probe cleavage coupled with isothermal amplification.

The outbreak of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been challenging human health worldwide. Loop-mediated isothermal amplification (LAMP) has been promptly applied to the detection of SARS-CoV-2 owing to its high amplification efficacy and less requirement of the thermal cycler. However, the vast majority of these LAMP-based assays depend on the non-specific detection of LAMP products, which can not discern the undesirable amplificons, likely to yield unreliable results. Herein, a sequence-specific LAMP assay was reported to detect SARS-CoV-2 using proofreading enzyme-mediated probe cleavage (named Proofman), which could realize real-time and visual detection without uncapping. This assay, introducing a proofreading enzyme and the fluorogenic probe to reverse-transcription LAMP (RT-Proofman-LAMP), can specifically detect the SARS-CoV-2 RNA with a detection limit of 100 copies. In addition to the real-time analysis, the assay is capable of endpoint visualization under a transilluminator within 50 min, providing a convenient reporting manner under the setting of point-of-care testing (POCT). In combination with different fluorophores, the one-pot multiplex assay was successfully achieved to detect multiple targets of SARS-CoV-2 and inner control simultaneously. In summary, the development of RT-Proofman-LAMP offers a versatile and highly-specific method for fast field screening and laboratory testing of SARS-CoV-2, making it a promising platform in COVID-19 diagnosis.

Long non-coding RNA PWRN2 regulates cytotoxicity in an in vitro model of age-related macular degeneration.

BACKGROUND: Age-related macular degeneration (AMD) may lead to irreversibly vision loss among aging populations. In this work, in an in vitro AMD cell model, we examined the expression and function of long non-coding RNA, Prader-Willi Region Non-Protein Coding RNA 2 (PWRN2) in injured human retinal pigment epithelial cells. METHOD: ARPE-19 cell line was maintained in vitro and treated with multi-module stressful conditions, including hydrogen peroxide (H2O2) tert-butylhydroperoxide (t-BuOOH) and ultraviolet B (UVB). Multi-module-stressor-induced cell death was monitored by a viability assay, and PWRN2 expression by qRT-PCR. PWRN2 was either downregulated or upregulated in ARPE-19 cells. The effects of PWRN2 downregulation or upregulation on t-BuOOH-induced cell death, cellular apoptosis and mitochondrial injuries were then quantitatively evaluated. RESULTS: Multi-module stressful conditions induced cell death and PWRN2 upregulation in ARPE-19 cells in vitro. We created ARPE-19 subpopulations with either downregulated or upregulated PWRN2 expressions. Quantitative assays demonstrated that, PWRN2 downregulation effectively alleviated t-BuOOH-induced cell death, apoptosis and various-type of mitochondrial injuries. On the other hand, PWRN2 upregulation worsened t-BuOOH-induced cellular damages in ARPE-19 cells. CONCLUSION: We demonstrated that downregulating PWRN2 protected multi-module-stressor-induced cell death, apoptosis and mitochondrial injuries in human retinal pigment epithelial cells, suggesting PWRN2 may be an active factor in human AMD.

Wenyang Lishui Decoction Ameliorates Podocyte Injury in Membranous Nephropathy Rat and Cell Models by Regulating p53 and Bcl-2.

Wenyang Lishui decoction (WYD) has been frequently used to treat patients with membranous nephropathy (MN) in China. Our previous study in vitro showed that WYD aqueous extract could alleviate F-actin reorganization of podocytes induced by serum from idiopathic membranous nephropathy (IMN) patients. This study aims to investigate the effects and molecular mechanisms of WYD on MN. MN rat models were induced by cationic bovine serum albumin. Experimental rats were divided into four groups: normal, model, WYD, and benazepril. The normal group consisted of normal rats receiving distilled water for four weeks, while the model, WYD, and benazepril groups consisted of MN rats receiving distilled water, 16.5 g/kg/day WYD aqueous extract, and 10 mg/kg/day benazepril, respectively. Alanine aminotransferase, kidney function, albumin, and 24 h urine total protein (UTP) were measured. Hematoxylin-eosin and electron microscopy analyses were performed. Mouse podocytes were induced to develop cell models by serum from IMN patients with antibody to the M-type phospholipase A2 receptor and spleen and kidney Yang deficiency syndrome. They were divided into five groups: control, model, 2 mg/ml WYD, 4 mg/ml WYD, and 8 mg/ml WYD. CCK-8 assays, flow cytometry, qRT-PCR, and Western blot analyses were performed. In the animal experiment, side effects of WYD were not found. Also, there was no significant difference in kidney function among the groups. In addition, UTP level was significantly reduced, and kidney histological damage was restored in both WYD and benazepril groups but difference in UTP level between them was not found. In the cell experiment, apoptosis rate was increased in the model group while it was decreased by coincubation with WYD. Besides, mRNA and protein levels of p53 were decreased, and those of Bcl-2 were increased by treatment using WYD. In conclusion, WYD could reduce proteinuria and ameliorate podocyte injury by regulating the expression of p53 and Bcl-2. The study is registered in the Chinese Clinical Trial Registry (ChiCTR-OCH-14005137).

Intracellular Selection of Theophylline-Sensitive Hammerhead Aptazyme.

Hammerhead ribozyme-based aptazyme (HHAz), inheriting the advantages of small size and high efficiency from the RNA-cleaving ribozyme and the specific recognition ability of aptamers to specific targets, exhibits the huge potential to be a transgene expression regulator. Herein, we report a selection strategy for HHAz by using a toxin protein IbsC as the reporter to offer a positive phenotype, thus realizing an easy-operating, time- and labor-saving selection of HHAz variants with desired properties. Based on this strategy, we obtained a new HHAz (TAP-1), which could react sensitively toward the extracellular regulatory molecule, theophylline, both in prokaryotic and eukaryotic systems. With fluorescent protein reporter, the intracellular switching efficiencies of TAP-1 and other reported theophylline-dependent HHAzs has been quantitatively evaluated, showing that TAP-1 not only exhibits the best downregulating ability at high concentration of theophylline but also maintains high activity with 0.1 mM theophylline, which is a safe concentration in the human body.

Frame-shifted proteins of a given gene retain the same function.

Frameshift mutations are generally considered to be lethal because it could result in radical changes of the protein sequence behind. However, the protein of frameshift mutants of a type I toxin (ibsc) was found to be still toxic to bacteria, retaining the similar function as wild-type protein to arrest the cellular growth by impairing the membrane's integrity. Additionally, we have verified that this observation is not an individual event as the same phenomenon had been found in other toxins subsequently. After analyzing the coding sequence of these genes, we proposed a hypothesis to search this kind of hidden gene, through which a dihydrofolate reductase-encoding gene (dfrB3) was found out. Like the wild-type reductase, both +1 and -1 frame-shifted proteins of dfrB3 gene were also proved to catalyze the reduction of dihydrofolate to tetrahydrofolate by using NADPH.